Bactericidal/permeability-increasing protein (BPI) compositions

ABSTRACT

Bactericidal/permeability increasing protein (BPI) and biologically active fragments, analogs and variants thereof are solubilized by lipid carriers including phospholipids, liposomes and nonionic detergents and stabilized against particle formation by poloxamer surfactants.

BACKGROUND OF THE INVENTION

The present invention relates to the solubilization and stabilization ofbactericidal/permeability-increasing protein (BPI) and biologicallyactive polypeptide fragments, analogs and variants thereof. Moreparticularly, the invention provides compositions of BPI and suchBPI-related polypeptides with lipids and, particularly, lipid carriersubstances such as phospholipids, liposomes and nonionic detergents aswell as compositions with poloxamer surfactants. Also provided areimproved pharmaceutical compositions for use as parenteral drugs.

Recent advances in the development of genetic engineering technologyhave made a wide variety of biologically active polypeptides availablein sufficiently large quantities for use as drugs. Recombinant BPI andfragments, analogs and variants of BPI, like other polypeptides, can besubject to particulate formation and loss of biological activity by avariety of chemical and physical means including denaturation due toheating or freezing and exposure to extreme pH or other chemicaldegradation. Particulate formation and loss of biological activity canoccur as a result of physical agitation and interactions of polypeptidemolecules in solution and at the liquid-air interfaces during theprocess of isolation and upon storage within vials. It is believed thatthe polypeptide molecules adsorb to an air-liquid interface, unfoldingto present hydrophobic groups to air with the hydrophilic groupsimmersed in the aqueous phase. Once so positioned at the surface, thepolypeptide molecules are susceptible to aggregation, particle formationand precipitation. It is also believed that further conformationalchanges can occur in polypeptides adsorbed to air-liquid andsolid-liquid interfaces during compression-extension of the interfacessuch as occurs from agitation during compression-extension of theinterfaces such as occurs from agitation during transportation orotherwise. Such agitation can cause the protein to entangle, aggregate,form particles and ultimately precipitate with other adsorbed proteins.Particle formation due to surface denaturation can be somewhatcontrolled by appropriate selection of the dimensions of storage vialsand by minimizing the air volume (headspace) in those vials. In thisregard, partially filled containers represent the worst case forvibration induced precipitation.

Particle formation has traditionally been controlled by incorporation ofsurfactants into the protein-containing composition in order to lowerthe surface tension at the solution-air interface. Classic stabilizationof pharmaceuticals by surfactants or emulsifiers (e.g., lipid carriers)has focused on the amphipathic nature of molecular groups containingboth hydrophilic and hydrophobic properties within the surfactantmolecule.

Wang et al., J. Parenteral Sci. & Technol., 42, supp. 25, pp. S4-S26(1988) review the use of surfactants as stabilizers for proteins andpeptides in parenteral drug formulations. Specifically, polysorbate 20and polysorbate 80 are cited as stabilizers for pharmaceuticalcompositions including those containing interleukin-2, tissueplasminogen activator and tumor necrosis factor. Wang et al. furtherdisclose the use of BRIJ surfactants for the stabilization ofpharmaceutical compositions comprising uricase and insulin and the useof an otherwise uncharacterized poloxamer surfactant for thestabilization of an insulin composition. Of interest to the presentinvention is the work related to use of polysorbate 80 ( TWEEN 80),poloxamer-188 (PLURONIC F-68) and steareth-100 (BRU 700) forstabilization of antibody-based product formulations as described inLevine, et al., J. Parenteral Sci. Technol., 45, 3, 160-165 (1991). Amonoclonal antibody product, OKT3 (Ortho Pharmaceutical Corp.) approvedby U.S. regulatory authorities for human use is formulated withpolysorbate 80.

Although a variety of surfactants or emulsifiers have been used tosolubilize/stabilize polypeptide compositions, regulatory requirementslimit the types and specific identities of surfactants that can beincorporated into parenteral compositions for injection into the humanbody. Generally accepted surfactants having a history of use and listedin the U.S. Pharmacopoeia XXII include polysorbate (polyoxyethylenesorbitol ester) and poloxamer (polyoxypropylene-polyoxyethylene blockcopolymer) polymers. Polysorbate 80 has been approved in parenteralsolutions for over 20 years, but is rarely used in concentrationsgreater than 0.1% in solution volumes of 100 mL or more. For example,Krantz et al., "Sugar Alcohols - XXVIII. Toxicologic, Pharmacodynamicand Clinical Observations on TWEEN 80," Bull. of the School of Med., U.of MD., 36, 48 (1951), identifies the onset of hemolysis in the dog fora polysorbate concentration of 0.1% at 90 minutes. In addition, neonataldeaths have been associated with the use of polysorbate 80 atconcentrations of greater than 1%. With respect to poloxamersurfactants, the highest safe concentration for poloxamer 188 inapproved parenteral solutions was 2.7% in an approved parenteral useblood substitute perfluorochemical solution where it was diluted as muchas 10 fold in the bloodstream. Thus, certain concentrations ofsurfactants may pose increased risk of toxic effects, earlier onset ofhemolysis, and observed changes in both neutrophils and platelets, whichare involved in blood complement activation.

BPI and fragments, analogs and variants of BPI are susceptible toparticulate formation and loss of biological activity. Nevertheless, theart has failed to show means for the stabilization of such BPIpolypeptides or for the solubilization of BPI polypeptide aggregates,particles or precipitates. Accordingly, there exists a need in the artfor BPI compositions providing improved protein solubilization andstability. Moreover, there exists a need in the art for pharmaceuticalcompositions comprising BPI or biologically active fragments, analogs orvariants thereof which comprise only concentrations of components whichare regarded as safe and are included in parenterals approved byregulatory authorities for commercial use.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising abactericidal/permeability-increasing protein (BPI) or a biologicallyactive polypeptide fragment, analog or variant thereof (produced byrecombinant or nonrecombinant means) and a lipid carrier, particularly anonionic detergent lipid carrier, where the BPI polypeptide issolubilized in the lipid carrier. Useful nonionic detergent lipidcarriers include octoxynol-9 (TRITON X-100, Rohm & Haas), polysorbate 80(TWEEN 80, ICI Americas, Inc., Wilmington Del.), polysorbate 20 TWEEN20, ICI Americas, Inc.) and laureth-4 (BRU 30, ICI Americas, Inc.). Thenonionic detergent lipid carriers, such as polysorbate 80solubilize/stabilize BPI polypeptides by altering (generally lowering)the surface tension of the polypeptide solution. The invention alsoprovides a method of solubilizing/stabilizing such BPI polypeptides bycontacting the polypeptide with a lipid carrier under conditions suchthat the polypeptide is solubilized.

The invention additionally relates to the discovery that a poloxamersurfactant is particularly useful for the solubilization/stabilizationof compositions comprising an aqueous solution of BPI protein orbiologically active fragments, analogs, or variants of BPI protein. Theinvention provides a method of solubilizing/stabilizing suchpolypeptides without altering the surface tension by contacting thepolypeptide solution with a poloxamer surfactant, for example, poloxamer188. The poloxamer surfactant component is preferably present in aconcentration of from about 0.01% to about 1% by weight with aconcentration of 0.1% to 0.2% by weight being preferred to stabilizeprotein solutions comprising less than or equal to 2 mg/mL.

The invention further relates to compositions comprising abactericidal/permeability-increasing protein (BPI) or a biologicallyactive polypeptide fragment, analog or variant thereof and aphospholipid or liposome lipid carrier wherein the polypeptide issolubilized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 graphically represent the results of bacterial growthinhibition assays for rBPI₂₃ with and without polysorbate 20 surfactantaddition.

FIG. 3 depicts the results of a Limulus Amoebocyte Lysate (LAL) assayusing rBPI₂₃ formulated in polysorbate 20.

FIG. 4 is a graph depicting surface tension measurements of rBPI₂₁solutions with varying surfactant concentrations of polysorbate 80(PS80) and poloxamer 188 (F68).

FIG. 5 is a series of graphs of differential scanning calorimetryresults of rBPI₂₁ with various concentrations of the surfactantpoloxamer 188 (F68).

FIG. 6 is another series of graphs of differential scanning calorimetryresults of rBPI₂₁ with various concentrations of poloxamer 188 (F68).

FIG. 7 is a plot showing the change in denaturation and precipitationtemperatures of rBPI₂₁ over varying concentrations of the surfactantpoloxamer 188 (F68).

FIG. 8 is a series of graphs of differential scanning calorimetryresults of rBPI₂₁ with various concentrations of polysorbate 80 (PS80)alone or in combination with 0.1% poloxarner 188 (F68) by weight.

FIG. 9 is a set of graphs of differential scanning calorimetry resultsof rBPI₂₁ with the surfactant polysorbate 80 (PS80) at two differentconcentrations.

FIG. 10 is a set of graphs of differential scanning calorimetry resultsafter a solution of rBPI₂₁ and poloxamer 188 (F68) was heated to atemperature higher than the denaturation/unfolding temperature but lowerthan the precipitation temperature, and then was cooled for repeatscanning.

DETAILED DESCRIPTION

The present invention provides methods and materials for maintaining thesolubility/stability of BPI polypeptide compositions with lipidcarriers, particularly nonionic detergent lipid carriers, phospholipidand liposome lipid carriers, and with poloxamer surfactants. Theinvention further provides methods and materials for maintaining thesolubility/stability of BPI polypeptide pharmaceutical compositionsusing only concentrations of components which are regarded as safe andare included in parenterals approved by regulatory authorities forcommercial use. Specifically, the invention relates to the discoverythat nonionic detergent molecules provide improvements in solubilizationand stabilization of BPI and biologically active fragments, analogs andvariants thereof. The invention also relates to the discovery thatpoloxamer surfactants have unique properties in thesolubilization/stabilization of such BPI-related proteins. It isbelieved that poloxamer surfactants protect BPI and fragments, analogsand variants thereof from surface denaturation by stabilizing unfoldedand partially unfolded BPI polypeptide molecules and preventingprecipitation of those molecules, and not by altering the surfacetension of the BPI solution.

BPI and biologically active fragments, analogs and variants thereofuseful with the present invention include recombinantly producedproteins such as described e.g., in U.S. Pat. No. 5,198,541. Co-owned,copending patent application Theofan et al., U.S. patent applicationSer. No. 08/064,693 filed May 19, 1993, which is a continuation-in-partapplication of U.S. Ser. No. 07/885,911 filed May 19, 1992, abandonedaddresses BPI-Immunoglobulin fusion proteins which are variants of BPIprotein comprising at the amino terminal a BPI protein or a biologicallyactive fragment thereof, and retaining the same biological activity ofBPI protein. The disclosures of these patent applications areincorporated by reference herein. Particularly preferred BPI analogmaterials include recombinant polypeptides produced according toco-owned and copending Theofan et al. U.S. patent application Ser. No.08/013,801 filed Feb. 2, 1993, now U.S. Pat. No. 5,420,019 and entitled"Stable Bactericidal/Permeability-Increasing Protein Products andPharmaceutical Compositions Containing the Same," the disclosure ofwhich is herein incorporated by reference. A preferred BPI fragment ischaracterized by about 1 to 199 or about 1 to 193 of the amino-terminalamino acid residues of the mature human BPI molecule as set out in Grayet al., J. Biol. Chem., 264, 9505-9509 (1989) except that residue 185 isglutamic acid rather than lysine as specified in Gray. The recombinantexpression product of DNA encoding BPI amino acids 1 to 199 has beendesignated rBPI₂₃. The recombinant expression product of DNA encodingBPI amino acids 1 to 193 has been designated rBPI(1-193). A preferredBPI fragment analog comprises the first 193 amino acid residues as setout in Gray except that residue 185 is glutamic acid rather than lysineand the cysteine at position 132 is replaced with a non-cysteine residuesuch as alanine. Such a protein is designated rBPI₂₁ orrBPI(1-193)ala¹³².

The present invention provides a composition comprising BPI andbiologically active fragments, analogs and variants thereof and a lipidcarrier. A pharmaceutically acceptable lipid carrier is preferred foruse in medicinal products designed for internal use.

As used in this application a lipid carrier is any fat soluble substancewhich inhibits protein precipitation. Lipid carriers may comprisesterile solutions and gels. Compositions comprising such lipid carriersare formulated by well known conventional methods.

The lipid carrier may be a phospholipid, a liposome, alipopolysaccharide (such as bacterial endotoxin), a fatty acid or adetergent. As used herein, a detergent is any substance that alters thesurface tension of a liquid, generally lowering it. The detergent may bea nonionic detergent. Examples of nonionic detergents includeoctoxynol-9 (TRITON X-100, Rohm & Haas), polysorbate 80 (TWEEN 80, ICIAmericas, Inc., Wilmington Del.), polysorbate 20 (TWEEN 20, ICIAmericas, Inc.) and laureth-4 (BRIJ 30, ICI Americas, Inc.). Preferrednonionic detergents are polysorbate 80 and polysorbate 20.

A preferred polysorbate nonionic detergent preferably has a surfacetension between 10 and 70 mN/m as measured in aqueous solution at roomtemperature and at a concentration of 0.1%. More preferably, thepolysorbate is characterized by a hydrophilic/lipophilic balance (HLB)value of about 15 and by a surface tension between 40 and 50 mN/m asmeasured in aqueous solution at room temperature and at a concentrationof 0.1%. Most preferred is polysorbate 80 (sorbitanmono-9-octadeconoate) which is available commercially as TWEEN 80 (ICIAmericas, Inc.).

Poloxamer surfactants are members of the group ofpolyoxypropylene-polyoxyethylene block copolymers and are characterizedby having generally hydrophilic properties. Poloxamer surfactants arebelieved to promote solubilization/stabilization of BPI and fragments,analogs and variants thereof by a mechanism that does not involvealtering the surface tension of the BPI polypeptide solution. Poloxymersurfactants are believed to protect the polypeptide from surfacedenaturation by stabilizing unfolded and partially unfolded BPIpolypeptide molecules.

Preferred poloxamer surfactants are characterized by a HLB value greaterthan about 14 and a surface tension between 10 and 70 mN/m as measuredin aqueous solution at room temperature and at a concentration of 0.1%.More preferred is a poloxamer surfactant which has a HLB value betweenabout 25 and 35 and has a surface tension between 30 and 52 mN/m asmeasured in aqueous solution at room temperature and at a concentrationof 0.1%. Most preferred is poloxamer 188 available commercially asPLURONIC F-68 (BASF Wyandotte, Parsippany, N.J.), which is characterizedby a surface tension of 50 mN/m and by an HLB value of 29.

The invention will be better understood upon consideration of thefollowing illustrative examples of practice thereof wherein: Example 1addresses solubilization of solutions of recombinant products with avariety of materials including phospholipids, liposomes and nonionicdetergents; Example 2 addresses biological activities of recombinantproducts in compositions with polysorbate 20; Examples 3 and 4 addresstests of various surfactants to determine their utility for surfacestabilization of BPI₂₃ ; Example 5 addresses evaluation of the surfaceactivity of polysorbate and poloxamer surfactants alone or in solutionsof rBPI₂₁ ; and Example 6 addresses Differential Scanning Calorimetry(DSC) analysis of rBPI₂₁ solutions prepared with polysorbate andpoloxamer surfactants.

EXAMPLE 1

In this example, the effects of various methodologies for stabilizingsolutions of recombinant BPI proteins produced by transfected CHO cellswere examined. It was found that when rBPI₂₃ was eluted from S-Sepharosebeads with a sodium chloride/acetate buffer, the resulting BPIpreparation contained significant amounts of hairy, thread-like orfilamentous precipitates. Addition of nonionic detergents to the bufferused to elute rBPI₂₃ solubilized the protein and prevented formation ofthe hairy, thread-like precipitates, but increased the quantities ofother contaminants eluted. Addition of nonionic detergents to elutedrBPI₂₃ preparations containing the hairy, thread-like precipitatesdecreased the size and number of the precipitates. Phospholipids weremixed with an rBPI₂₃ solution and a solution of full-length recombinantBPI was sonicated with liposome kit materials. Elution of rBPI₂₃directly into a solution comprising a nonionic detergent preventedformation of the thread-like precipitates and avoided elution of othercontaminants from the cell culture medium.

1(A). Eluting rBPI₂₃ with Buffer Containing Nonionic Detergents

In this experiment, different methods were used to elute rBPI₂₃ fromS-Sepharose beads that had been cultured in roller bottles with CHOcells transfected with DNA encoding the 31-residue signal sequence andamino acids 1 to 199 of mature BPI protein. The procedures were carriedout according to the general method of Example 1 of co-owned andco-pending U.S. patent application Ser. No. 08/072,063 filed May 19,1993 which is a continuation-in-part of U.S. patent application Ser. No.07/885,501, the disclosures of which are hereby incorporated byreference. See also, PCT WO 93/23540 published Nov. 25, 1993.Specifically, S-Sepharose beads from five roller bottles of transfectedCHO cells were separated from cells and culture media with a milliporefilter and washed with 20 mM sodium acetate/acetic acid, 100 mM NaCl pH4.0 (Buffer). The washed beads were resuspended into 15 mL fractions andeach fraction was treated with a different eluting solution. The elutingsolutions were (1) Buffer with 0.7 M and 1.0 M NaCl; (2) 0.1%polysorbate 20 in Buffer with 0.7 M, 1.0 M and 1.5 M NaCl; (3) 0.1%polysorbate 80 in Buffer with 0.7 M, 1.0 M and 1.5 M NaCl; (4) 0.5%octoxynol-9 (TRITON X-100) in Buffer with 1.0 M NaCl; and (5) a taurineisopropanol solution with 0.35 M, 0.7 M and 1.0 M NaCl.

The eluate obtained using Buffer and sodium chloride alone (No. 1,above) contained significant amounts of hairy, thread-like precipitates.Polysorbate 20, polysorbate 80, octoxynol-9 (TRITON X-100), and taurineprotected BPI from formation of thread-like precipitates on elution.These eluates, however, also contained increased quantities of culturemedium contaminants.

1(B). Treatment of rBPI₂₃ Samples with Nonionic Detergents and LipidCarriers After Elution

Samples of rBPI₂₃ protein solutions containing thread-like precipitateswere contacted with a variety of agents to assess the ability of theagent to dissipate or dissolve the precipitate. In one experiment, twomL aliquots of the solutions were placed in 15 mL screw cap tubes. Thevarious agents, including phospholipid, TWEEN 80, TWEEN 20 and TRITONX-100, were added, mixed by hand, and visually compared to control(untreated) samples. The results are set out in Table 1, which reflectsthe approximate concentration of the agent added. Agents which appearedto prompt formation of additional thread-like precipitates are indicatedby the letter A; agents having little apparent effect are indicated bythe letter B; the letter C indicates agents which appeared to dissipatethe thread-like precipitates but resulted in cloudy solutions with smallprecipitates; and the letter D indicates agents that decreased thenumber and size of the thread-like precipitates. The addition of 0.1%TWEEN 20 to the BPI₂₃ solution provided the best effects. In asubsequent experiment 0.1% TWEEN 20 alone did not appear to dissipatethe thread-like precipitate and 1% deoxycholate appeared to promptprecipitate formation.

                  TABLE 1    ______________________________________    AGENT                  RESULT    ______________________________________    100 μL phospholipid (Culture PL, AH Labs)                           A    10 μL endotoxin suspension                           A    3M thiocyanate         A    2% taurine             A    0.05% octoxynol-9 (TRITON X-100)                           A    2% lysine              A    10% dimethylsulfoxide  A    2% albumin             A    20% glycerol           B    2% glycine             B    2 mM ethylenediamine tetra acetic acid                           B    5% polyethylene glycol C    30% acetic acid        C    60% acetonitrile       C    2% sodium dodecyl sulfate                           C    5% isopropanol         D    10% ethanol            D    0.1% polysorbate 80 (TWEEN 80)                           D    0.1% polysorbate 20 (TWEEN 20)                           D    ______________________________________

An attempt was made to use liposome materials to dissipate thread-likeprecipitates in a solution of the recombinant expression product of aDNA sequence encoding the entire 456 amino acid residues of mature humanBPI. The solution was sonicated with a lipid mixture (liposome kit,Sigma Chemical, St. Louis, Mo.) with little apparent effect on theprecipitates.

1C. Elution into a Nonionic Detergent Solution

Washed beads according to Example 1A were eluted with Buffer into a tubewhich contained polysorbate 20 such that the final concentration ofpolysorbate 20 in the solution was 0.01%. The elution of sample directlyinto the polysorbate 20 solution appeared to drastically reduce theamount of hairy, thread-like precipitates in the preparation.

EXAMPLE 2

In this example, naturally-occurring BPI holoprotein and recombinant BPIfragments, analogs and variants thereof were subjected to assays todetermine the effect of polysorbate 20 nonionic detergent on thebiological activity of the protein. Compositions of such products withpolysorbate 20 were variously tested in E. coli growth inhibitionassays, in an in vitro Limulus Amoebocyte lysate inhibition assay and inan in vivo LPS toxic challenge assay. The results showed thatincorporation of polysorbate 20 nonionic detergent in the formulationshad no detrimental effect on the biological activity in the assays.

2(A). E. Coli Growth Inhibition Assays

In this example, E. coli growth inhibition assays were conducted todetermine the effect of polysorbate 20 on the biological activities ofrBPI₂₃, naturally-occurring BPI holoprotein, rBPI₂₁ andrBPI-immunoglobulin fusion proteins according to Theofan et al. U.S.application Ser. Nos. 07/885,911 filed May 19, 1992 and 08/064,693 filedMay 19, 1993. The results for combinations of polysorbate 20 with rBPI₂₃set out below are representative of the results for the other BPIpolypeptides and demonstrate the absence of any adverse effect bypolysorbate 20 on rBPI₂₃ polypeptide. Broth assays were performed tocompare the effect of formulations of rBPI₂₃ with 0.1% polysorbate 20nonionic detergent (TWEEN 20) and formulations of rBPI₂₃ withoutpolysorbate 20 on the growth of E. coli J5, a rough UDP-galactose 4epimerase negative mutant of the smooth E. coli strain 0111-B4. In eachassay, cells were grown overnight in TYE broth Gazzano-Santoro et al.,Infect. Immun., 60: 4754 (1992)! and then subcultured in triethanolaminebuffered minimum salt medium TEA, Weiss et al., J. Clin. Invest., 65:619 (1980)!. The bacterial cells were harvested in late-logarithmicphase, centrifuged and resuspended in 0.9% NaCl to provide a suspensionof about 5×10⁸ cells/mL (A₆₀₀ ˜0.5). Aliquots of the suspensionproviding about 10⁷ cells were added to 0.4 mL of suspension medium 10mL comprising 1.0 mL Hanks Balanced Salts Solution, 0.4 mL/M Tris-HCl(pH 7.5), 0.2 mL of 5% Casamino acids, 1.0 mL 9% NaCl and water tovolume!. 20 μl buffered (20 mM Sodium Acetate, pH 4, 1.0 M NaCl) rBPI₂₃test solutions at a concentration of 1 μg/mL and/or buffer controls wereadded to the cells, which were then incubated at 37° C. for 30 minutes.Dilutions of formulated rBPI₂₃ to 1 μg/ml were performed using buffereither with or without polysorbate 20 as indicated in Table 2 below.Four mL of nutrient broth Difco Laboratories Inc., Detroit, Mich., 0.9%NaCl! was added to each sample. Tubes were incubated on a rotary shakerand read at A₆₀₀ at 2, 4 and 6 hours with the results shown in FIG. 1wherein assay (1) is represented by the solid square; assay (2) isrepresented by the solid circle; assay (3) is represented by the solidtriangle; assay (4) which overlaps with assay (5) is represented by anopen square; and assay (5) which overlaps with assay (4) is representedby an open circle. The results of this experiment show that rBPI₂₃ inthe presence of polysorbate 20 actually had greater cell growthinhibition activity than rBPI₂₃ in buffer without the nonionicdetergent.

                  TABLE 2    ______________________________________    Additives to Bacterial Cell Suspension                                    Final BPI    Assay No.           BPI Preparation                         BPI Diluent                                    Conc. (μg/ml)    ______________________________________    1      rBPI.sub.23 in Acetate                         Acetate Buffer                                    1           Buffer    2      rBPI.sub.23 in 0.1%                         0.1%       1           TWEEN.sup.1   TWEEN.sup.1    3      rBPI.sub.23 in 0.1%                         Acetate Buffer                                    1           TWEEN.sup.1    4      Acetate Buffer                         NA         0    5      0.1% TWEEN.sup.1                         NA         0    ______________________________________     .sup.1 Acetate buffer containing 0.1% TWEEN 20.

In a second series of experimental assays the rBPI₂₃ compositions setout in Table 3 below were tested in the same assay described above withthe results shown in FIG. 2 wherein assay 1 is represented by an opensquare; assay 2 is represented by an open diamond; assay 3 isrepresented by an open circle; assay 4 is represented by an opentriangle; assay 5 is represented by an open square containing a plussign; and assay 6 is represented by an open diamond containing a plussign. Results of assays 4, 5 and 6 are largely overlapping. The resultsof this experiment show that rBPI₂₃ in the presence of polysorbate 20actually had greater cell growth inhibition activity than rBPI₂₃ inbuffer without the nonionic detergent.

                  TABLE 3    ______________________________________    Additive to Bacterial Cell Suspension                                      Final BPI    Assay No.            BPI Preparation                           BPI Diluent                                      Conc. (μg/ml)    ______________________________________    1       rBPI.sub.23 in Acetate Buffer                           Acetate Buffer                                      1    2       rBPI.sub.23 in Acetate Buffer                           Acetate    1                           Buffer.sup.1    3       rBPI.sub.23 in 0.1%                           0.1%       1            TWEEN.sup.2    TWEEN    4       Acetate Buffer only                           NA.sup.3   0    5       0.1% TWEEN.sup.2 only                           NA.sup.3   0    6       Buffer only    NA.sup.1,3 0    ______________________________________     .sup.1 2 μl of 1% TWEEN added directly to cells in suspension medium.     .sup.2 Acetate buffer containing 0.1% TWEEN 20.     .sup.3 Not applicable.

2(B). In Vitro LAL Inhibition Assay

Solutions comprising rBPI₂₃ were subjected to a Limulus AmoebocyteLysate (LAL) inhibition assay to determine the effect of polysorbate 20nonionic detergent on the LPS binding properties of rBPI₂₃.Specifically, an rBPI₂₃ preparation was obtained by elution with a 20 mMsodium acetate/acetic acid buffer at pH 4.0 and 0.7 M to 1.0 M NaCl fromS-Sepharose beads cultured in roller bottles containing transfected CHOcells. The preparation was then (1) treated with polysorbate 20 (TWEEN20) to a concentration of 0.05% (closed triangles in FIG. 3); (2)treated with sodium azide to a concentration of 0.02% (open circles inFIG. 3) or (3) left untreated (open diamonds in FIG. 3). A second rBPI₂₃preparation comprising 0.4 mg/mL in 10 mM potassium phosphate pH 7.0,0.15 M NaCl and 0.05% polysorbate 20 (closed squares in FIG. 3) was alsoused in the assay. The BPI preparations were mixed in Eppendorf tubeswith a fixed oncentration of E. coli 0113 LPS and incubated at 37° C.with occasional shaking. control with no LPS (open squares in FIG. 3)was also tested. Following incubation, D-PBS was added to each tube toreduce the LPS concentration for the LAL assay. Each sample was thentransferred into Immulon II strips (Dynatech, Chantilly, Va.) in volumesof 50 μl per well.

Limulus amoebocyte Lysate (Quantitative chromogenic LAL kit, WhitakerBioproducts, Inc., Walkersville, Md.) was added to each well accordingto the kit instructions and the wells were incubated at roomtemperature. Chromogenic substrate was then added to each well and wasmixed well with the contents. After incubation at room temperature, thereaction was stopped with addition of 25% acetic acid. Optical densityat 405 nm was then measured in a multiplate reader (Vmax, MolecularDynamics, Menlo Park, Calif.) with the results shown in FIG. 3.Inhibition of LPS activation of LAL by rBPI₂₃ is indicated by theconcentration-dependent drop in OD₄₀₅.

The results show that the inclusion of polysorbate 20 had no detrimentaleffect on the biological activity of rBPI₂₃ in the assay.

2(C). In Vivo LPS Toxic Challenge Assay

In this example, a study was conducted to assess the efficacy of rBPI₂₃formulated with polysorbate 20 nonionic detergent lipid carrier in anactinomycin-D sensitized mouse model according to Pieroni et al., Proc.Soc. Exp. Biol. & Med.; 133, 790 (1970). ICR mice were administered anintravenous injection of actinomycin-D (800 μg/kg). Immediatelythereafter, a group of 15 mice received injections of 100 μg/kg E. coli0111:B4 LPS and 3.8 mg/kg rBPI₂₃ in a composition comprising 10 mMpotassium phosphate 0.17 M NaCl pH 4.0 and 0.05% polysorbate 20. Ascontrols, some animals were treated with buffer but not BPI and otheranimals were treated with actinomycin D but not with the LPS or BPI.

The results presented in Table 4 show that no deaths occurred in hecontrol animals not treated with LPS and that fewer deaths occurred inthe animals treated with rBPI₂₃ formulated with polysorbate 20 (13/15)than in the animals treated with buffer (15/15).

                  TABLE 4    ______________________________________    E. coli 0.111:B4                Act-D       rBPI.sub.23                                     No. Dead/    μg/kg    800 μg/kg                            3.8 mg/kg                                     Total    ______________________________________     0          +           -         0/15    100         +           -        15/15    100         +           +        13/15    ______________________________________

EXAMPLE 3

In this example, tests of various surfactants were conducted todetermine their utility for surface stabilization of a recombinant BPIfragment polypeptide pharmaceutical (rBPI₂₃). The rBPI₂₃ was provided ata concentration of 1 mg/mL in citrate buffered saline (0.02 M citrate,0.15 M NaCl, pH 5.0). Various surfactants were then added to thispreparation in order to determine their utility as stabilizers.

According to this test, rBPI₂₃ was filled by hand to 5 mL in sealedsterile 5 mL molded glass vials (total capacity 8.4 mL, Wheaton) in thedesired formulation buffer. The vials to be tested were set horizontallyon a flat bed shaker (S/P rotor V) and fixed to the shaker by tape.Vials were then shaken at 150 rpm at room temperature. At 0 hours, 2-4hours, and 18 hours, 150 μl samples were withdrawn in a biosafetycabinet using a 1 mL syringe fitted with a 21 gauge needle. Thestarting, in process, and ending soluble rBPI₂₃ concentrations weredetermined by an ion exchange HPLC assay and visual observation ofcloudiness of the solution was also recorded. The results are shownbelow in Table 6 in which acceptable stability was determined by visualinspection after the shake test.

Testing of protein preparations comprising single surfactants showedgood results for use of octoxynol-9 (TRITON X-100, Rohm & Haas),laureth-4, (BRIJ 30, ICI Americas), poloxamer 403 (PLURONIC P123, BASFWyandotte) and telomere B monoether with polyethylene glycol (ZONYLFSO-100, E.I. DuPont de Nemours).

Testing of other surfactants as shown in Table 5 shows that surfactantsproducing a surface tension lower than 35 mN/m are capable ofstabilizing rBPI at surfactant concentrations of 0.1%. This examplefurther shows that both polysorbate 80 (TWEEN 80) and poloxamer 188(PLURONIC F-68) were incapable of stabilizing the protein preparationalone under the shake test conditions employed. The incorporation ofpolysorbate 80 did, however, have the effect of clarifying a cloudysolution of BRIJ 30 which is not readily water soluble without the helpof an additional solubilizer.

                                      TABLE 5    __________________________________________________________________________              Surface Tension              mN/m at 0.1% Conc.                        Surfactant         rBPI.sub.23 Conc. by HPLC    Exp       Surfactant              at Room Temp. in                        Concentration in                                Visual Observation                                           (mg/mL)    No.       Used   Water (w), Buffer (b).sup.1                        Form. Buffer                                3-4 hr.                                     18 hr 0 hr                                              3-4 hr                                                  18 hr    __________________________________________________________________________    1  ZONYL  17.sup.(w)                        0.100%  --   Clear 0.96                                              --  1.00       FSO-100    2  PS-80  41.sup.(b)                        0.100%  --   Cloudy                                           1.11                                              --  0.02    3  BRIJ 30              27.5.sup.(b)                        0.500%  Cloudy                                     Cloudy.sup.(2)                                           1.08                                              --  1.14    4  TRITON 32.sup.(b)                        0.100%  Clear                                     Clear 1.00                                              1.01                                                  0.98       X-100    5  PLUR P123              34.3.sup.(w)                        0.100%  Clear                                     Clear 1.08                                              1.08                                                  1.08    6  BRIJ 30/PS-80              --        0.1%/   Clear                                     Clear 1.19                                              1.21                                                  1.17                        0.125%    7  PLUR   46.sup.(b)                        0.100%  Clear                                     Haze; 1.23                                              1.22                                                  0.95       F-68                          specks.    8  PLUR   44.sup.(b)                        0.200%  Clear                                     Haze  -- --  1.04       F-68                          with a                                     few                                     specks.    __________________________________________________________________________     .sup.1 Surface tensions with superscript w are obtained from the     surfactant manufacturer. Surface tensions with superscript b are obtained     experimentally using Wilhelny plate method.     .sup.2 Brij 30 alone is cloudy.

EXAMPLE 4

In this example, additional comparisons were carried out according tothe methods of Example 3 using various surfactants alone to stabilize arBPI₂₃ preparation. The results are shown below in Table 6 in whichacceptable stability was determined by visual inspection after the shaketest.

                                      TABLE 6    __________________________________________________________________________               Surfactant    Exp        Conc. in Form.                      Visual Observation                                 Conc. by HPLC (mg/mL)    No.       Surfactant Used               Buffer 3-4 hr                           18 hr 0 hr                                     3-4 hr                                          18 hr    __________________________________________________________________________     1 ZONYL    0.100%                      --   Clear 0.96                                     --   1.00       FSO-100     2 PS-80    0.100%                      --   Cloudy                                 1.11                                     --   0.02     3 Dextran Sulfate                1 mg/mL                      --   Cloudy                                 --  --   0.00     4 Glycerol               10.0%  --   Cloudy                                 0.86                                     --   0.02     5 HSA      5.0%  --   Cloudy                                 0.92                                     --   0.00     6 Control-               --     --   Cloudy                                 1.13                                     --   0.03       5 mL Fill       Volume     7 Control --     --   Clear. One                                 1.13                                     --   1.04       8.4 mL              speck of       (complete)          precipitate.       Fill: Volume     8 Control-               --     Cloudy                           Cloudy                                 1.16                                     0.21 0.00       5 mL (partial)       Fill Volume     9 TRITON   0.500%                      Clear                           Clear 1.04                                     0.99 1.11       X-100    10 PS-80    0.500%                      Clear                           Cloudy                                 1.12                                     0.95 0.59    11 PLURONIC                0.500%                      Clear                           Clear 1.15                                     --   1.13       P123    12 BRIJ 30  0.500%                      Cloudy.sup.(1)                           Cloudy.sup.(1)                                 1.08                                     --   1.14    13 TRITON   0.100%                      Clear                           Clear 1.00                                     1.01 0.98       X-100    14 TRITON   0.010%                      Slt. Haze                           Cloudy                                 0.96                                     0.84 0.04       X-100    15 PLURONIC                0.100%                      Clear                           Clear 1.08                                     1.08 1.08       P123    16 PLURONIC                0.100%                      Clear                           Clear 1.23                                     1.26 0.94       P123    17 PLURONIC                0.050%                      Clear                           Slt. Haze                                 1.21                                     1.18 1.11       P123    18 PLURONIC                0.010%                      Cloudy                           Cloudy                                 1.14                                     0.06 0.00       P123    19 BRIJ 30/                0.1%/ Clear                           Clear 1.19                                     1.21 1.17       PS-80    0.125%    20 BRIJ 30/                0.075%/                      Clear                           Clear 1.22                                     1.20 1.18       PS-80    0.094%    21 BRIJ 30/                0.03%/                      Slt. Haze                           Cloudy                                 1.20                                     1.05 0.41       PS-80    0.038%    22 BRIJ 30/                0.01%/                      Cloudy                           Cloudy                                 1.14                                     0.48 0.00       PS-80    0.013%    23 PLURONIC                0.100%                      Clear                           Slt. Haze                                 1.23                                     1.22 0.95       F68    24 PLURONIC                0.100%                      Clear                           Slt. Haze                                 --  --   1.00       F68    25 PLURONIC                0.150%                      Clear                           Slt. Haze                                 --  --   1.06       F68    26 PLURONIC                0.200%                      Clear                           Slt. Haze                                 --  --   1.04       F68    27 PLURONIC                0.300%                      Clear                           Slt Haze                                 --  --   1.10       F68    28 PLURONIC                0.500%                      Clear                           Slt. Haze                                 --  --   1.08       F68    29 PLURONIC                0.070%                      Clear                           Clear 1.06                                     1.08 0.97       P123    30 BRIJ 30/                0.05%/                      Clear                           Clear 1.04                                     1.01 1.01       PS-80    0.063%    31 PLURONIC                0.100%                      Cloudy                           Cloudy                                 1.07                                     0.87 0.56       F88    32 PLURONIC                0.100%                      Cloudy                           Cloudy                                 1.04                                     0.77 0.39       F98    33 PLURONIC                0.100%                      Clear                           Cloudy                                 1.04                                     0.87 0.55       F108    34 PLURONIC                0.100%                      Clear                           Clear 1.06                                     1.04 0.98       F127    35 PLURONIC                0.100%                      Clear                           Clear 1.12                                     --   0.93       F127    36 PLURONIC                0.075%                      Clear                           Clear 1.10                                     --   0.61       F127    37 PLURONIC                0.050%                      Clear                           Slt. Haze                                 1.09                                     --   0.20       F127    38 PLURONIC                0.025%                      Slt. Haze                           Cloudy                                 1.07                                     --   0.00       F127    39 PLURONIC                0.010%                      Cloudy                           Cloudy                                 1.06                                     --   0.00       F127    __________________________________________________________________________     .sup.(1) Brij 30 alone is cloudy.

EXAMPLE 5

In this example surface tension measurements were made of polysorbateand poloxamer surfactants alone, or combinations of the two in solutionsof the BPI protein product rBPI₂₁ according to the procedure set out inthe Kruss Digital Tensiometer K10ST Users Manual, Chapter 4: Measuringwith the Plate. A decrease in surface tension indicates an increase inthe surface activity of the surfactant, which has conventionally beenthought to be the mechanism by which surfactants stabilize proteins.These procedures established that poloxamer surfactants provideadvantageous results by a different and unexpected mechanism.

Specifically, a 2 mg/mL solution of unformulated rBPI₂₁ (lot 30216) wasdiluted with 20 mM sodium citrate, 150 mM sodium chloride, pH 5.0rendering a 1 mg/mL solution. 15 mL of this solution was placed into a50 mL glass beaker containing a mini stir bar. Surfactants poloxamer188, polysorbate 80, or combinations of both were added incrementally upto 0.10%. Before each surface tension measurement, the platinum platewas heated above the reducing zone (blue flame) of a gas burner untilthe plate just began to glow red. The platinum plate was heated forabout 10 to 15 seconds while turning the plate from side to side andthen suspended back into the instrument. Each addition of surfactant wasgently mixed using a magnetic stirrer and the solution was allowed tostand for 2 minutes on the thermostat vessel equilibrated at 4.6° C. Thevalue for the surface tension was read after five minutes.

The first part of this experiment evaluated the surface activity of thesurfactants alone in buffer. Using the citrate saline buffer (20 mMsodium citrate, 150 mM sodium chloride, pH 5.0) as the baseline,surfactants were added incrementally. FIG. 4 is a plot of surfacetension dependence on surfactant concentrations; the corresponding datais presented in Table 7. The open squares represent the citrate salinebuffer in varying concentrations of poloxamer 188 while the closedcircles represent the same buffer in varying concentrations ofpolysorbate 80. The citrate-saline buffer solution alone had a surfacetension of about 75 mN/m at 4.6° C., similar to H₂ O. With increasingconcentrations of surfactants, the buffer solution showed decreasingsurface tension. With 0.10% poloxamer 188, the surface tension of thesolution was 55 mN/m. On the other hand, with 0.10% polysorbate 80, thesurface tension of the solution was 45 mN/m. The decrease in surfacetension indicates an increase in the surface activity of the surfactant,i.e., the lower the surface tension, the higher the surface activity.The results indicate that polysorbate 80 is more surface active thanpoloxamer 188.

In the second part of the experiment, the surface activity of rBPI₂₁ inthe presence of surfactants was evaluated. The results show that rBPI₂₁at 1 mg/mL in citrate saline buffer, pH 5.0, is surface active with asurface tension of about 54 mN/m at 4.6° C. The addition of polysorbate80 (PS80) alone up to 0.0005% did not change the surface tension ofrBPI₂₁ solution either (FIG. 4, closed triangles). At concentrations ofpolysorbate 80 exceeding 0.0005%, the surface tension of rBPI₂₁ followsthat of buffer with PS80 alone (no BPI), in which the surface tension ofthe solution decreases as the concentration of polysorbate 80 isgradually increased. For buffer with PS80 alone, the surface tension of54 mN/m was reached when the PS80 concentration was increased from0.0005%. These results indicate that when PS80 concentration is lessthan 0.0005%, the surface activity of the solution is dominated byrBPI₂₁. On the other hand, at PS80 concentrations above 0.0005%, thesurface activity of the solution is modulated by polysorbate 80. Theaddition of poloxamer 188 (F68) alone to rBPI₂₁ at concentrations up to0.10% did not change the surface activity of rBPI₂₁ solutionsignificantly (FIG. 4, open triangles). Addition of varyingconcentrations of polysorbate 80 to an rBPI₂₁ solution containing 0.1%poloxamer 188 is represented by the closed squares in FIG. 4.

                                      TABLE 7    __________________________________________________________________________                                  8          2       4       6       rBPI.sub.21                                            10          Buffer  Buffer  rBPI.sub.21                                  + 0.1% F68                                            rBPI.sub.21    1     + F68              3   + PS80                      5   + F68                              7   + PS80                                        9   + PS80    % F68 (mN/m)              % PS80                  (mN/m)                      % F68                          (mN/m)                              % PS80                                  (mN/m)                                        % PS80                                            (mN/m)    __________________________________________________________________________     1      0.00000          75.4              0.00000                  75.1                      0.00000                          54.2                              0.00000                                  53.7  0.00000                                            54.9     2      0.00001          74.9              0.00001                  66.8                      0.00001                          54.7                              0.00001                                  53.4  0.00001                                            55.0     3      0.00003          74.3              0.00002                  60.0                      0.00002                          54.2                              0.00002                                  53.3  0.00002                                            53.2     4      0.00005          68.2              0.00003                  60.0                      0.00003                          54.9                              0.00003                                  53.9  0.00003                                            53.3     5      0.00007          65.9              0.00005                  60.0                      0.00004                          54.8                              0.00004                                  53.9  0.00004                                            52.8     6      0.00010          64.0              0.00007                  57.4                      0.00005                          55.0                              0.00005                                  53.5  0.00005                                            52.4     7      0.00013          65.8              0.00010                  56.6                      0.00006                          55.2                              0.00006                                  53.5  0.00006                                            53.3     8      0.00015          65.4              0.00015                  57.2                      0.00007                          55.4                              0.00007                                  53.4  0.00007                                            53.6     9      0.00017          66.5              0.00020                  56.7                      0.00008                          54.8                              0.00008                                  53.8  0.00008                                            53.8    10      0.00020          65.7              0.00050                  55.6                      0.00009                          55.0                              0.00010                                  53.4  0.00009                                            53.2    11      0.00023          66.0              0.00070                  55.3                      0.00010                          54.9                              0.00020                                  53.5  0.00010                                            53.5    12      0.00027          64.4              0.00100                  54.2                      0.00030                          55.3                              0.00030                                  53.2  0.00020                                            53.2    13      0.00030          63.8              0.00300                  52.7                      0.00050                          54.5                              0.00050                                  52.3  0.00030                                            53.0    14      0.00033          64.1              0.00700                  49.2                      0.00070                          55.5                              0.00070                                  51.5  0.00050                                            52.0    15      0.00037          63.1              0.01000                  48.3                      0.00100                          54.9                              0.00100                                  51.0  0.00070                                            51.2    16      0.00040          64.2              0.03000                  46.5                      0.00500                          54.9                              0.00200                                  50.6  0.00100                                            50.5    17      0.00043          61.8              0.07000                  45.3                      0.01000                          55.4                              0.00500                                  50.1  0.00130                                            50.4    18      0.00047          62.4              0.10000                  45.4                      0.05000                          53.6                              0.01000                                  48.6  0.00170                                            49.8    19      0.00050          63.1        0.10000                          53.7                              0.05000                                  45.6  0.00200                                            48.8    20      0.00060          61.6                0.10000                                  45.0  0.00500                                            47.7    21      0.00070          62.5                          0.01000                                            46.7    22      0.00080          62.0                          0.05000                                            45.4    23      0.00100          61.7                          0.10000                                            45.0    24      0.00300          61.2    25      0.00500          59.3    26      0.00700          58.9    27      0.01000          58.4    28      0.03000          56.6    29      0.07000          56.1    30      0.10000          55.1    __________________________________________________________________________

EXAMPLE 6

Protein samples were analyzed by Differential Scanning Calorimetry (DSC)to study the unfolding (or denaturation) of the protein. The startingmaterials for DSC analysis were identical to those used in the surfacetension measurement. A series of rBPI₂₁ solutions was prepared withvarying concentrations of surfactants, poloxamer 188, polysorbate 80 orcombinations of both, and diluted with buffer (20 mM sodium citrate, 150mM sodium chloride, pH 5.0) to give a final rBPI₂₁ concentration of 1mg/mL. A series of buffer solutions was also prepared with surfactantsat the same concentrations as in the rBPI₂₁ solutions to serve as blanksfor DSC. Each solution was filtered and placed into a 2 mL sterileplastic vial. The samples were packed into a 4° C. cold box untilsubjected to DSC analysis.

The behavior of rBPI₂₁ was evaluated as the temperature of the solutionwas gradually increased from ambient temperature to about 90° C., at arate of 1° C. per minute. As the temperature is increased two eventsoccur. The first event is an unfolding reaction, which is endothermic,and is illustrated by an upward peak in the scans. The second event isprecipitation, which is exothermic, and is depicted by a downward peakin the scans. In the scans depicted in FIGS. 5, 6 and 8-10, each scan isoffset to facilitate analysis of data. In the rBPI₂₁ solution notcontaining surfactants (FIG. 5, Scan 1) the unfolding of the protein at65° C. was followed immediately by the second event, precipitation ofthe protein at 66 to 67° C.

With low poloxamer 188 (PLURONIC® F68) concentrations ranging between0.001% to 0.01%, the unfolding and precipitation events are similar tothe rBPI₂₁ solution without surfactants (FIG. 5, Scans 2 to 5), i.e. asrBPI₂₁ unfolds, precipitation takes place immediately. With poloxamer188 concentrations exceeding 0.05%, the unfolding of rBPI₂₁ still occursat 65° C., but precipitation does not occur until the temperaturereaches 85° C. (FIG. 5, Scan 6). FIG. 6 shows that at poloxamer 188concentrations between 0.01% and 0.05%, there is a gradual transition ofdelayed precipitation of unfolded BPI. These results suggest that atpoloxamer 188 concentrations higher than 0.01%, unfolded rBPI₂₁ can bestabilized and the occurrence of precipitation is delayed. A plot ofdenaturation and precipitation temperature dependence over varyingpoloxamer 188 concentration is shown in FIG. 7. The effects of poloxamer188 appear to delay the precipitation of rBPI₂₁ to a higher temperaturebut not to stabilize its native structure as the T_(m) (denaturationtemperature) and ΔH (energy of denaturation) did not change.

rBPI₂₁ formulated with polysorbate 80 at concentrations up to 1% waslikewise analyzed by DSC (FIG. 8: Scans 1 and 8-13, FIG. 9: Scans 11,12). The isotherms were similar to rBPI₂₁ solution without surfactants.Polysorbate 80 did not maintain the rBPI₂₁ in solution at highertemperatures. The stabilization of unfolded rBPI₂₁ is thus unique topoloxamer 188.

The two formulations using combined poloxamer 188 and polysorbate 80,namely 0.1%F68/0.001%PS80 and 0.1%F68/0.002%PS80 (FIG. 8, Scans 14, 15),showed the same scan profile as rBPI₂₁ containing 0.05% and 0.1%PLURONIC F68, with unfolding at 65° C. and precipitation at 85° C. (FIG.6, Scans 6, 7).

In addition to determining the melting behavior of rBPI₂₁, rescanningwas done with rBPI₂₁ formulations containing 0.05% and 0.10% poloxamer188 to determine if unfolding is a reversible process. The temperatureof the rBPI₂₁ solution was first increased to 75° C. (temperature afterdenaturation/unfolding but before precipitation), then was cooled downfor repeat scanning. FIG. 10 shows that the addition of poloxamer 188 torBPI₂₁ does not make unfolding reversible. Profiles A5,1 and A6,1 showthe scanning to 75° C., while profile A5,2 and A6,2 are repeat scanningafter cooling the system from 75° C. If unfolding were a reversibleprocess, scan profiles 6 and 7 would have been obtained.

The experimental results described above demonstrate that poloxamersurfactant alone is capable of stabilizing BPI-related polypeptides insolution and delaying the occurrence of precipitation by a mechanismthat does not appear to involve modulation of the surface tension of theaqueous solution. This property is unique to poloxamer because othersurfactants which are detergents such as polysorbate 80 affect theprecipitation phenomenon by modulation of the surface tension of theaqueous solution.

Numerous modifications and variations of the above-described inventionare expected to occur to those of skill in the art. Accordingly, onlysuch limitations as appear in the appended claims should be placedthereon.

What is claimed is:
 1. A composition comprising abactericidal/permeability-increasing protein (BPI) or a biologicallyactive BPI fragment or BPI analog thereof and a lipid carrier, whereinthe lipid carrier is a nonionic detergent, and wherein thebactericidal/permeability-increasing protein or biologically active BPIfragment or BPI analog thereof is solubilized in the lipid carrier. 2.The composition of claim 1 wherein the nonionic detergent is polysorbate80.
 3. A composition comprising a bactericidal/permeability-increasingprotein or biologically active BPI fragment or BPI analog thereof and aliposome or phospholipid lipid carrier wherein thebactericidal/permeability-increasing protein or biologically active BPIfragment or BPI analog thereof is solubilized in the lipid carrier.